The RT-PCR reaction setup usually requires the reaction tube to be opened after the cDNA synthesis step for the addition of PCR reaction components, increasing the risk of contamination. The Titan One Tube RT-PCR System is designed for fast, sensitive and reproducible analysis of RNA by high fidelity RT-PCR in a one-step reaction. In this system, both cDNA synthesis and PCR are combined in one reaction tube using an optimized buffer, eliminating the need to add further reagents between these two steps. It uses AMV reverse transcriptase for first strand cDNA synthesis and the Expand High Fidelity enzyme blend (consisting of Taq and Tgo DNA polymerases) for amplification of cDNA by PCR. Tgo DNA polymerase possesses proofreading activity leading to an increased PCR fidelity. The system includes a single optimized RT-PCR buffer.
Titan claims that the One Tube RT-PCR System reduces the error rate in PCR due to the proofreading capability. A three-fold higher fidelity may be obtained as compared to Taq DNA polymerase. It is not yet known whether there is also an increase in fidelity during the reverse transcription part of RT-PCR. Titan also claims that this system allows the amplification of fragments up to 6 kb by RT-PCR due to the use of the Expand PCR System, as well as, delivering increased sensitivity related to the high efficiency of all three enzymes. Finally, Titan asserts that this kit is faster than conventional two-step RT-PCR.
All reactions are performed in UV-crosslinked, thin-walled PCR tubes. The reactions require the formation of two “Master Mixes” and take approximately thirty minutes to an hour to set up. The first Master Mix contains double-distilled water, dNTPs, downstream and upstream primers, template RNA (1ug-1pg), DTT solution and Protector RNase Inhibitor. The second Master Mix also has double distilled water, but includes the 5x RT-PCR buffer with Mg2+ and the enzyme mix. You combine 25 ul of the first Master Mix with 25 ul of the second in a thin walled tube, place it on ice and then put the tube into a thermocycler equilibrated at 50 C for 30 minutes, following the program outlined in the kit instruction manual.
We use the Titan One Tube RT-PCR in two different capacities in our research, which falls under the broad category of genetic engineering. We must first identify possible gene targets for up-regulation, and, once found, we must clone these genes into cells and observe the effects. The Titan One Tube RT-PCR kit aids us first in helping to confirm the relative RNA expression between samples of our target gene through amplification using primers targeting for that gene. We also use the same kit to amplify the copy number of a gene so that we can purify it from that PCR reaction and clone it into an expression vector. Our lab has been using the Titan One Tube RT-PCR System for some time now and we can not do without it. The procedure is fast and the results come out accurate and precise with nice, clear bands on our gels. Our complaints about the product stem from the manual. While it does give a thorough explanation on how the system works, it does not list many troubleshooting points. I would recommend this kit for the experienced PCR technician, who already has a basic knowledge of how the procedure works and can troubleshoot on their own without any problem.
Andrea Smiley
Research Specialist II
Shriner’s Hospital for Children, Cincinnati